Examinando por Autor "Barros, M. José"

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    A feed-forward loop between SroC and MgrR small RNAs modulates the expression of eptB and the susceptibility to polymyxin B in Salmonella Typhimurium
    (Microbiology Society, 2016-11) Acuña, Lillian G.; Barros, M. José; Peñaloza, Diego; Rodas, Paula I.; Paredes-Sabja, Daniel; Fuentes, Juan A.; Gil, Fernando; Calderón, Iván L.
    Base-pairing small RNAs (sRNAs) regulate gene expression commonly by direct interaction with cognate mRNAs. Nevertheless, recent studies have expanded this knowledge with the discovery of the RNA ‘sponges’ which are able to interact and repress the functions of classical base-pairing sRNAs. In this work, we present evidence indicating that the sponge RNA SroC from Salmonella enterica serovar Typhimurium base pairs with the MgrR sRNA, thereby antagonizing its regulatory effects on both gene expression and resistance to the antimicrobial peptide polymyxin B (PMB). By a predictive algorithm, we determined putative SroC–MgrR base-pairing regions flanking the interaction area between MgrR and its target mRNA, eptB, encoding a LPS-modifying enzyme. With a two-plasmid system and compensatory mutations, we confirmed that SroC directly interacts and down-regulates the levels of MgrR, thus relieving the MgrR-mediated repression of eptB mRNA. Since it was previously shown that an Escherichia coli strain carrying an mgrR deletion is more resistant to PMB, we assessed the significance of SroC in the susceptibility of S. Typhimurium to PMB. Whereas the sroC deletion increased the sensitivity to PMB, as compared to the wild-type, the resistance phenotypes between the DmgrR and DsroCDmgrR strains were comparable, evidencing that mgrR mutation is epistatic to the sroC mutation. Together, these results indicate that both SroC and MgrR sRNAs compose a coherent feed-forward loop controlling the eptB expression and hence the LPS modification in S. Typhimurium.
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    Detection of Nucleic Acids of the Fish Pathogen Yersinia ruckeri from Planktonic and Biofilm Samples with a CRISPR/Cas13a-Based Assay
    (Multidisciplinary Digital Publishing Institute (MDPI), 2024-02-02) Calderón, Iván L.; Barros, M. José; Fernández-Navarro, Nicolás; Acuña, Lillian G.
    Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.